Compositions and methods for treating diseases by inhibiting exosome release

ABSTRACT

A multipartite peptide that inhibits release of exosomes in a cell, comprising an N-terminal end and a C-terminal end and comprising at least one secretion modifying region (SMR) peptide from HIV-1 Nef and at least one Clusterin (Clu)-binding peptide (Clu-BP). Pharmaceutical compositions comprising these peptides alone or in synergistic combinations with other active agents in methods for treating cancers and/or infectious diseases are further provided herein.

This application is a Continuation of U.S. application Ser. No.15/383,454, filed Dec. 19, 2016. The entirety of the aforementionedapplications is incorporated herein by reference.

FIELD

The present disclosure generally relates to compositions and methods formedical treatment, and in particular, to methods for treating cancersand infectious diseases by inhibiting exosome release.

BACKGROUND

Membrane exosomes are spherical membrane microvesicles, generally lessthan 200 nm in diameter. The exosomes are composed of a lipid bilayercontaining a cytosolic fraction. Particular membrane vesicles are morespecifically produced by cells, from intracellular compartments throughfusion with the cytoplasmic membrane of a cell, resulting in theirrelease into the extracellular biological fluids of an organism or intothe supernatant of cells in culture. These exosomes may be released in anumber of ways. The classical secretory pathway processes mainlytraditional membrane signals bearing receptors through the endoplasmicReticulum (ER) membrane (Lee et al., (2004) Annu. Rev. Cell Dev. Biol.20, 87-123).

Secretory proteins are packaged into transport vesicles, delivered tothe Golgi apparatus, and eventually released of into the extracellularspace. Alternatively, nonclassical secretory pathways mediatetranslocation of cytosolic, nonsignal bearing molecules into theextracellular space (Lippincott-Schwartz et al., (1989) Cell 56,801-813; and Misumi et al., (1986) J. Biol. Chem. 261, 11398-11403). Twoof these involve intracellular vesicles of the endocytic membranesystem, such as secretory lysosomes (Muesch et al., (1990) TrendsBiochem. Sci. 15, 86-88) and exosomes (Johnstone et al., (1987) J. Biol.Chem. 262, 9412-9420), the latter ones being internal vesicles of lateendosomes or multivesicular bodies (MVB). Lysosomal contents gain accessto the exterior of cells when specialized endocytic structures such assecretory lysosomes of cytotoxic T lymphocytes, fuse with the plasmamembrane. Lumenal contents of late endocytic structures are releasedinto the extracellular space when MVBs fuse with the plasma membraneresulting in release of the internal multivesicular endosomes into theextracellular space (called exosomes) along with their cargo molecules.Other nonclassical pathways involve direct translocation of cytosolicfactors across the plasma membrane using protein conducting channels ora process called membrane blebbing (Nickel, W. (2005) Traffic. 6,607-614). Membrane blebbing is characterized by shedding of plasmamembrane-derived microvesicles into the extracellular space.

Exosome release has been demonstrated from different cell types invaried physiological contexts. It has been demonstrated that tumor cellssecrete exosomes, such as exosomes in a regulated manner, which cancarry tumor antigens that can be presented to antigen presenting cells(Patent Application No. WO99/03499). In addition, FasL or TNF containingexosomes are known to cause a state of immune privilege/immunesuppression which can promote tumor growth. Similarly, virus-infectedcells, including those infected by HIV are known to releaseNef-containing exosomes (Guy et al., (1990) Virology 176, 413-425; andCampbell et al., (2008) Ethn. Dis. 18, S2-S9), which serve to suppressthe immune system allowing HIV to survive. Exosome secretion has beenshown to utilize the same endosomal trafficking pathway involved invirion release from infected cells (Sanfridson et al., (1997) Proc.Natl. Acad. Sci. U.S.A 94, 873-878; and Esser et al., (2001) J Virol.75, 6173-6182).

Tumors are known to release large numbers of exosomes, which can causeimmune suppression through immune cell killing or dysregulation, therebypromoting a state of immunosuppression that allows for rapid tumorgrowth (Lindner K. et al., 2015, Salido-Guadarrama I. et al., 2014).Similarly, HIV infections result in high numbers of exosomes, whichappears to contribute to a state of immune privilege/suppression whichultimately could lead to Acquired Immune Deficiency Syndrome (AIDS).

The exosome secretion pathway serves a dual function in both regulationof the cancer homeostasis, the immune system and virion release ofinfected cells. In view of the foregoing, there is a need in the art forcompositions and effective methods of treatment for inhibiting exosomerelease.

SUMMARY

One aspect of the present disclosure relates to a multipartite peptidethat inhibits the release of exosomes from cells. The peptide containsat least one secretion modifying region (SMR) peptide from HIV-1 Nef andat least one Clusterin (Clu)-binding peptide (Clu-BP).

In one embodiment, the peptide contains at least one SMR peptidesequence, such as VGFPV (SEQ ID NO: 1) or VGFPVAAVGFPV (SEQ ID NO:2),and at least one Clu-BP peptide sequence selected from the groupconsisting of HPLSKHPYWSQP (SEQ ID NO:3), NTYWSQLLHFQT (SEQ ID NO:4) andSHALPLTWSTAA (SEQ ID NO:5). In one embodiment, the peptide has an SMRpeptide motif at its N-terminal end and a Clu-BP peptide motif at itsC-terminal end. In another embodiment, the peptide has a Clu-BP peptideat its N-terminal end and an SMR peptide at its C-terminal end.

In certain embodiments, the peptide has a plurality of SMR peptidemotifs separated by a suitable spacer peptide, a plurality of Clu-BPpeptide motifs separated by a suitable spacer peptide, or both.

In a particular embodiment, the peptide comprises an amino acid sequenceselected from the group consisting of VGFPVAAVGFPVHPLSKHPYWSQP (SEQ IDNO:6), VGFPVAAVGFPVAAHPLSKHPYWSQP (SEQ ID NO:7),VGFPVAAVGFPVAAHPLSKHPYWSQPAAHPLSKHPYWSQP (SEQ ID NO:8).

In another embodiment, a pharmaceutical composition comprises amultipartite peptide in accordance with the present disclosure and apharmaceutically acceptable carrier.

Another aspect relates to polynucleotides and expression vectorsencoding the multipartite peptides described herein.

In a further aspect, a method for treating cancer or an infectiousdisease comprises administering to a subject in need of such treatmentan effective amount of a multipartite peptide in accordance with thepresent disclosure.

In another aspect, a method for treating AIDS comprises administering toa subject in need of such treatment an effective amount of amultipartite peptide in accordance with the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects and advantages of the application will beapparent upon consideration of the following detailed description, takenin conjunction with the accompanying figures and paragraphs. Thefollowing are brief descriptions of the drawings herein, whichillustrate certain aspects and embodiments of the present application,but are not considered limiting in any way.

FIG. 1 shows that SMRwt peptide antagonists inhibit proliferation ofMCF-7 and MDA-MB-231 breast cancer cells but not non-tumorigenic cells.Cells were incubated with peptides at varying dosage (0-1120 nM) for 24hr, after which proliferation was measured by MTT assay. Results ofthree independent experiments are shown. Panel A: Proliferation of MCF-7breast cancer cells. Panel B: Proliferation of MDA-MB-231 breast cancercells. Panel C: Proliferation of non-tumorigenic MCF-10A cells. Red dotsindicate PEG-SMRwt peptide, black dots indicate PEG-SMRwt-CLU peptide,and green triangles indicate PEG-SMRmut peptide.

FIG. 2 shows that PEG-SMRwt-CLU peptide antagonist and chemotherapeuticsinduced cell cycle arrest in MCF-7 and MDA-MB-231 breast cancer cells.Cells were treated 48 hr with SMR peptides, alone or in combination withpaclitaxel or cisplatin, and were assayed by Cellometer imagingcytometry, indicating percentage of MCF-7 (Panel A) and MDA-MB-231(Panel B) in various cell cycle phases. Results of two independentexperiments are shown. Significant differences relative to untreatedcontrol are indicated as follows: *p<0.01, **p<0.001 for MCF-7 cells,and *p<0.02, **p<0.01, ***p<0.0001 for MDA-MB-231 cells.

FIG. 3 shows that PEG-SMRwt-CLU peptide antagonist increasedcytotoxicity in MCF-7 but not in MDA-MB-231 breast cancer cells.Percentage of apoptotic cells (Panel A) MCF-7 and (Panel B) MDA-MB-231,as determined by Annexin V-FITC assay of cells treated for 48 hr withpeptide alone or combined with paclitaxel or cisplatin. Error barsrepresent mean±SD of four independent experiments. Significantdifferences relative to SMRwt peptide are indicated as follows: *p<0.01,***p<0.0001.

FIG. 4 shows that PEG-SMRwt-CLU peptide antagonist blocks exosomerelease from MCF-7 and MDA-MB-231 cells. Cells were treated for 48 hrwith peptide alone or combined with paclitaxel or cisplatin. Panels Aand B show relative level of exosomes released from MCF-7 and MDA-MB-231cells respectively, determined by AchE assay. Error bars representmean±SD of four independent experiments. Significant differencesrelative to SMRwt peptide: *p<0.01, **p<0.001, ***p<0.0001 for MCF-7cells; and *p<0.01 for MDA-MB-231 cells. Panels C and D show relativenumbers of exosomes released by MCF-7 and MDA-MB-231 cells respectively,as determined by Nanosight measurement. Error bars represent mean±SD oftwo independent experiments. Significant differences relative to SMRwtpeptide: *p<0.01, **p<0.001, ***p<0.0001 for MCF-7 cells and *p<0.03,**p<0.02, ***p<0.01 and ****p<0.001 on MDA-MB-231 cells.

FIG. 5 shows that exosome-specific proteins can be detected on exosomesfrom MCF-7 breast cancer cells. Cells were treated for 48 hr with SMRwtpeptide alone or combined with paclitaxel or cisplatin. Panel A:Expression of exosome proteins by Western blot analysis. Panel B:Exosome numbers were measured by NanoSight. Panel C: Densitometryanalysis showing relative intensity of bands. Data represent the mean±SDof three independent experiments. Significant differences relative totreatment with peptide are indicated as follows: *p<0.01, **p<0.001,***p<0.0001.

FIG. 6 shows that exosome-specific proteins can be detected on exosomesfrom MDA-MB-231 breast cancer cells. Cells were treated for 48 hr withSMRwt peptide alone or combined with paclitaxel or cisplatin. Panel Ashows Expression of exosome proteins by Western blot analysis. Exosomenumbers were measured by NanoSight (Panel B) and densitometry analysisshows relative intensity of bands (Panel C). Data represent the mean±SDof three independent experiments. Significant differences relative totreatment with peptide are indicated as follows: *p<0.01, **p<0.001,****p<0.0001.

FIG. 7 shows that antibody to mortalin inhibits exosome secretion fromMCF-7 breast cancer cells. MCF-7 cells were either transfected withantibodies to mortalin or alpha-tubulin, or treated with SMRwt or SMRmutpeptides. Panel A: Relative exosome release level after 48 hr by AchEassay. Panel B: Relative numbers of exosomes released after 48 hr byNanoSight analysis. Error bars represent the mean±SD of threeindependent experiments. Significant differences relative to untreatedcells: *p<0.0001, **p<0.0001.

FIG. 8 shows exosome secretion is decreased in MCF-7 breast cancer cellsby knockdown of mortalin expression. MCF-7 cells were transfected withclones expressing siRNA against either mortalin (HSPA9) or a negativecontrol RNA. Exosomes were isolated and analyzed after 24, 48, 72, and96 hr for changes in level of exosome secretion by AchE assay (Panel A).Significant differences relative to controls are indicated: ***p<0.0001,and exosome secretion by N-Rh-PE (Panel B). Significant differencesrelative to controls are indicated: ***p<0.0001. Panel C: percentage oflive cells remaining at each time point, *p<0.05, **p<0.002,***p<0.0001. Panel D: mortalin and CD63 protein expression levels byWestern blotting. Panel E: Densitometry analysis of Western blot data.Significant differences relative to controls are indicated:*p<0.01,**p<0.001,***p<0.0001.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed method and compositions belong. It must be notedthat as used herein and in the appended claims, the singular forms “a,”“an,” and “the” include plural reference unless the context clearlydictates otherwise. Thus, for example, reference to “a peptide” includes“one or more” peptides or a “plurality” of such peptides. With respectto the teachings in the present application, any issued patent or patentapplication publication described in this application is expresslyincorporated by reference herein. Further, where the phrases “in someembodiments . . . ” or “in certain embodiments . . . ” are used, thepresent disclosure should be construed as embracing combinations of anyof the features defining the different embodiments described herein,unless the features are not combinable with one another, are mutuallyexclusive, or are expressly disclaimed herein.

Definitions

As used herein, the terms “treat” and “treatment” refer to theamelioration of one or more symptoms associated with a disease, such ascancer or an infectious disease; prevention or delay of the onset of oneor more symptoms of the disease; and/or lessening of the severity orfrequency of one or more symptoms of the disease.

The term “cancer” refers to any one of a variety of malignant neoplasmscharacterized by the proliferation of cells that have the capability toinvade surrounding tissue and/or metastasize to new colonization sites,and includes leukemia, lymphoma, carcinoma, melanoma, sarcoma, germ celltumor and blastoma. Exemplary cancers for treatment with the methods ofthe instant disclosure include cancers of the brain, bladder, breast,cervix, colon, head and neck, kidney, lung, non-small cell lung,mesothelioma, ovary, prostate, stomach and uterus, leukemia, andmedulloblastoma.

The term “infectious diseases” includes those pathologic conditions thatarise from viruses, bacteria, fungi and/or parasites that invade anddisrupt the normal function of the mammalian body. Pages 3-147 of theMerck Manual 13th Edition describe some of these conditions and they areincorporated herein by reference.

The phrases “to a patient in need thereof”, “to a patient in need oftreatment” or “a subject in need of treatment” includes subjects, suchas mammalian subjects, that would benefit from administration of themultipartite peptide of the present disclosure for treatment of a cellproliferative disorder.

The terms “therapeutically effective amount”, “pharmacologicallyeffective amount”, and “physiologically effective amount” are usedinterchangeably to mean the amount of a multipartite peptide that isneeded to provide a threshold level of active antagonist agents in thebloodstream or in the target tissue. The precise amount will depend uponnumerous factors, e.g., the particular active agent, the components andphysical characteristics of the composition, intended patientpopulation, patient considerations, and the like, and can readily bedetermined by one skilled in the art, based upon the informationprovided herein or otherwise available in the relevant literature.

The terms, “improve”, “increase” or “reduce”, as used in this context,indicate values or parameters relative to a baseline measurement, suchas a measurement in the same individual prior to initiation of thetreatment described herein, or a measurement in a control individual (ormultiple control individuals) in the absence of the treatment describedherein.

A “control individual” is an individual afflicted with the same diseaseas the individual being treated, who is about the same age as theindividual being treated (to ensure that the stages of the disease inthe treated individual and the control individual(s) are comparable).The individual (also referred to as “patient” or “subject”) beingtreated may be a fetus, infant, child, adolescent, or adult human with acell proliferative disorder.

A “small molecule” refers to an organic or inorganic molecule that isnot a polymer, that has medicinal activity, and that has a molecularweight less than 1 kDa. The term encompasses most medicinal compoundstermed “drugs” other than protein or nucleic acids, although a smallmolecule peptide or nucleic acid analog can be considered a “smallmolecule”. Small molecules drugs can be derived synthetically,semi-synthetically (i.e., from naturally occurring precursors), orbiologically. As used herein, the phrase “large molecule” refers to apolymeric protein- or nucleic acid-based product having a molecularweight greater than 1 kDa.

Peptides

One aspect of the present disclosure relates to a multipartite peptidethat inhibits the release of exosomes from cells. In one embodiment, thepeptide comprises at least one secretion modifying region (SMR) peptidefrom HIV-1 Nef and at least one Clusterin (Clu)-binding peptide(Clu-BP).

In a particular embodiment, the SMR peptide comprises the amino acidsequence VGFPV (SEQ ID NO: 1) or VGFPVAAVGFPV (SEQ ID NO: 2), and atleast one Clu-BP peptide selected from the group consisting ofHPLSKHPYWSQP (SEQ ID NO:3), NTYWSQLLHFQT (SEQ ID NO:4) and SHALPLTWSTAA(SEQ ID NO:5) as described in U.S. Patent Publication No. 2012/0121507.

In one embodiment, the peptide comprises an SMR peptide at theN-terminal end and an Clu-BP peptide at the C-terminal end. In anotherembodiment, the peptide comprises a Clu-BP peptide at the N-terminal endand an SMR peptide at the C-terminal end.

In certain embodiments, the peptide comprises at least two SMR peptidesseparated by a spacer peptide, at least two Clu-BP peptides separated bya spacer peptide, or both.

In certain particular embodiments, the peptide comprises an amino acidsequence selected from the group consisting of VGFPVAAVGFPVHPLSKHPYWSQP(SEQ ID NO:6), VGFPVAAVGFPVAAHPLSKHPYWSQP (SEQ ID NO:7),VGFPVAAVGFPVAAHPLSKHPYWSQPAAHPLSKHPYWSQP (SEQ ID NO:8).

In certain embodiments, the multipartite peptide of the presentinvention further comprises one or more spacers between one or morefunctional domains within the multipartite peptide. The spacer isdesigned to facilitate the independent folding of each domain relativeto one another, ensure that the individual domains in the peptide do notinterfere with one another or with the SMR peptide and/or increase theflexibility of the protein and facilitate adoption of an extendedconformation. In some embodiments, the spacer comprises 1 to 50 aminoacids, preferably 2 to 10 amino acids.

In some embodiments, the spacer includes one or more a glycine and/orserine residues to force the spacer to adopt a loop conformation,because the absence of a (3-carbon permits the polypeptide backbone toaccess dihedral angles that are energetically forbidden for other aminoacids. In addition, spacers comprising glycine and/or serine have a highfreedom degree for linking of two peptides, i.e., they enable the fusedproteins to fold and produce functional proteins. Other residues thatcan enhance stability and folding include the amino acids alanine,proline, lysine, and combinations thereof. In one embodiment, the spaceris an Ala-Ala dipeptide linker. In another embodiment, the spacer hasthe formula [(Gly)_(n)-Ser/Ala]_(m) where n is from 1 to 4, inclusive,and m is from 1 to 4, inclusive.

In some embodiments, the multipartite peptide further comprises a cellpenetrating peptide (CPP) domain. A CPP domain enhances the uptake ofthe multipartite peptide into eukaryotic cells. Exemplary CPP domainsfor use in the present application include, but are not limited to, HIVTAT₄₉₋₅₇ peptide, HIV TAT₄₈₋₆₀ peptide, low molecular weight protamine(LMWP) peptide; Chariot™, also known as Pep-1 (Morris et al., Nat.Biotechnol., 19:1173-1176, 2001); Antp₄₃₋₅₈ peptide, MPG (HIV Gp41-SV40NLS), SAP, MPG R9, MAP, K-FGF, Penetratin, Buforin II, Transportan,Ku70, Prion, pVEC, Pep-1-K, Pep-7, HN-1, TP10, and CP26 (See e.g.,Joliot et al., Nature Cell Biol., 6(3):189-196, 2004 and Heitz et al.,Br. J. Pharmacol., 157:195-206, 2009).

In certain particular embodiments, the multipartite peptide furtherincludes a mitochondrial penetrating sequence or a mitochondrialtargeting signal sequence to facilitate uptake of the multipartitepeptides into the mitochondria where mortalin is localized. Exemplarymitochondrial targeting sequences include the presequence peptidedescribed in U.S. Patent Publication 2004/0192627, including thenuclear-encoded human cytochroine c oxidase (COX) subunit VIII(MSVLTPLLLRGLTGSARRLPVPRAKIHSL (SEQ ID NO:9)); the amino-terminal leaderpeptide of the rat ornithine transcarbamylase (OTC)(MLSNLRILLNKAALRKAHTSMVRNFRYGKPVQC (SEQ ID NO:10)), the presequence ofcytochrome oxidase subunit IV (MLSLRQSIRFFKPATRTL (SEQ ID NO:11)), andan Antennapedia α-helical domain, such as RQIKIWFQNRRMKWKK (SEQ IDNO:12); various mitochondrial targeting peptides described in U.S.Patent Publication No. 2014/0196172, including N-terminal mitochondrialtargeting peptides, MFSYLPRYPLRAASARALVRATRPSYRSALLRYQ (SEQ ID NO:13),MAAWMRSLFSPLKKLWIRMH (SEQ ID NO:14), MKLLWRLILSRKW (SEQ ID NO:15),MWWRRSRTNSLRYT (SEQ ID NO:16), and MLFRLRRSVRLRGLLA (SEQ ID NO:17); andthe N-terminal mitochondrial targeting peptide MWTLGRRAVAGLLASPSPAQ (SEQID NO:18) as described in U.S. Patent Publication No. 2016/0237129.Exemplary mitochondrial targeting signal peptide sequences directingproteins or peptides to the mitochondria include RRIVVLHGYGAVKEVLLNHK(SEQ ID NO:19), amino acids 74-95 of Rat Cytochrome P450 2E1 (CYP2E1),the cleavable prepiece from the yeast cytochrome c oxidase IV precursor(MLSLRQDIRFFKPATRTLCSSR (SEQ ID NO:20)), the mitochondrial-targetingsignal from the PB2 protein of influenza viruses, the import signalcontained within heme lyases, and the leader peptide of themitochondrial matrix enzyme ornithine transcarbamylase (OTC) asdescribed in U.S. Patent Publication No. 2014/0142121.

In some embodiments, the multipartite peptide may include a targetingdomain for targeting the peptide to specific types of cells, includingtumor cells, virally-infected cells and the like. The targeting domainmay comprise a peptide fused to the multipartite peptide or it may benon-peptide-based domain chemically conjugated to or covalently attachedthereto. Exemplary targeting domains include a peptides, smallmolecules, ligands, antibody fragments, and aptamers. In addition, atargeting domain may be a small molecule (e.g., folate, adenosine,purine) or a large molecule (e.g., peptide or antibody) thatspecifically binds to a desired target cell of interest. In someembodiments, the targeting domain is present at the C-terminal end ofthe multipartite peptide. In other embodiments, the targeting domain ispresent at the N-terminal end of the multipartite peptide.

In some embodiments, the multipartite peptide may be linked to animmunoglobulin Fc region. The Fc region can enhance stability and invivo half-life and can facilitate recruitment of Fc receptor-bearingnatural killer cells, macrophages, neutrophils, and mast cells, whichcan stimulate phagocytic or cytotoxic cells to destroy microbes orinfected cells by antibody-mediated phagocytosis or antibody-dependentcell-mediated cytotoxicity. When using antibody-derived targeting agentsor Fc regions, these domains are preferably “humanized” usingmethodologies well known to those of skill in the art.

The multipartite peptide encoded in the expression vector may furtherinclude a cleavage recognition sequence for proteolytic or endopeptidasecleavage sequence between one or more functional domains. Incorporationof endopeptidase cleavage recognition sequences can facilitate sitespecific cleavage by a suitable endopeptidase present in a eukaryotic ormammalian cell, such as asparagine endopeptidase, Factor Xa, furin,thrombin, cathepsin B, plasmin, and various matrix metalloproteinases(MMPs), such as MMP2, MMP7, MMP9, or MMP14. Placement of a suitableendopeptidase cleavage recognition sequence can serve to liberate anattached PEG moiety and/or liposomal moiety linked to the peptide, orliberate one or more peptide domains from one another so that one ormore these peptide domains can function independently of one another ine.g., their targeted site.

Asparagine endopeptidase, also known as legumain, is a lysosomalcysteine protease that cleaves protein substrates on the C-terminal sideof asparagine, such as Asn-Asp. Sequences cleavable by MMP2, MMP7, MMP9,or MMP14 include PLGLAG, PLG-C(me)-AG, RPLALWRS (SEQ ID NO:21), ESPAYYTA(SEQ ID NO:22), DPRSFL (SEQ ID NO:23), PPRSFL (SEQ ID NO:24), RLQLKL(SEQ ID NO:25), and RLQLK(Ac) (SEQ ID NO:26). Cathepsin B is a tumorassociated protease that can act upon the dipeptide sequencesvaline-citrulline and Phe-Lys. Furin cleaves the recongition sequenceArg-X-X-Arg (SEQ ID NO:27), more preferably Arg-X-(Lys/Arg)-Arg (SEQ IDNO:28). Factor Xa cleaves after the arginine residue in its preferredcleavage site Ile-(Glu or Asp)-Gly-Arg (SEQ ID NO:29) and will sometimescleave at other basic residues, depending on the conformation of theprotein substrate. The most common secondary site, among those that havebeen sequenced, is Gly-Arg. Thrombin preferentially cleaves between Argand Gly residues in e.g., the sequence LVPRGS (SEQ ID NO:30).

The multipartite peptide of the present disclosure may be chemicallymodified using one or more methods including, but not limited to,amidation, acetylation (including N-terminal acetylation),carboxylation, glycosylation, methylation (e.g., substitution ofα-hydrogens with methyl groups), carbonylation, phosphorylation,PEGylation, dimerization, addition of interchain and/or intrachaindisulfide bonds, addition of trans olefin, derivatization by knownprotecting/blocking groups, circularization, substitution with D aminoacids, linkage to an antibody molecules or other cellular ligands, etc.

The multipartite peptides of the present disclosure can be modified tocontain additional nonproteinaceous moieties that are known in the artand are readily available. Preferably, the moieties suitable forderivatization of the protein are water soluble polymers. Non-limitingexamples of water soluble polymers include, but are not limited to,polyethylene glycol (PEG), polyvinyl alcohol (PVA), copolymers ofethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol (PPG) homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol; POG), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water.

Additional modifications include, for example, point mutations,insertions, deletion, truncation, and backbone substitutions, such as NHto NCH₃, In addition, the peptide may be modified by the insertion ofone or more D amino acids. Further, proline analogs in which the ringsize of the proline residue is changed from 5 members to 4, 6, or 7members can be employed. Cyclic groups can be saturated or unsaturated,and if unsaturated, can be aromatic or non-aromatic.

The multipartite peptides may include modifications to, includingincorporation of any of the functional domains described herein at theN-terminal end of the peptide, the C-terminal end of the peptide, orboth. Alternatively, or in addition, modifications at the N-terminal endmay include an acetylated (Ac) residue and/or an amidated (NH₂) residueat the C-terminal end. Where the C-terminus is amidated, the carboxylicacid of the amino acid is converted to an amide, i.e., NH₂—CH₂—C(O)—NH₂.

The multipartite peptide may further contain one or more covalentlyattached functional groups, preferably attached to either or both of theN and C termini of the polypeptide. These covalently attached groups caninclude stabilizers, couplers, ligands, enzymatic substrates and/orcombinations thereof. Preferred groups include acyl groups on the Nterminus and cysteamine (cya) coupling groups on the C terminal end. Tothe latter may be conveniently attached other chemical moieties, e.g.,dyes, ligands, small molecule drugs, proteins, enzymes, enzymaticsubstrates, etc. Alternatives to cya are also known to those of skill inthe art. For stabilizing and/or blocking, e.g., cya may be replaced withan alky group such as methyl or ethyl, which are known to beconveniently positioned onto a —COOH group.

N-terminal modifications additionally may include, but are not limitedto, methylation (i.e., —NHCH₃ or —NH(CH₃)²), adding a1-amino-cyclohexane-carboxylic acid moiety (Chex); and adding acarbobenzoyl group, or blocking the amino terminus with any blockinggroup containing a carboxylate functionality defined by RCOO—, where Ris selected from the group consisting of naphthyl, acridinyl, steroidyl,and similar groups.

A derivitizing group, including, but not limited to, asulfhydryl-containing group or moiety may be positioned at theC-terminus of the multipartite peptide, even when it is not coupled toanother chemical moiety. In one embodiment, the C-terminal end may bemodified with a cysteamide group (—NH—CH₂—CH₂—SH), which can allowfurther coupling to drugs. A cysteamide group is compatible with thepeptide synthesis using the Fmoc strategy and leads to a C-terminalprotected peptide. Alternatively, the peptide can include a C-terminalcysteine residue containing a sulfhydryl (—SH) group that can beoptionally utilized for conjugation to other moieties. In anotherembodiment, the C-terminal end includes a 2,4-diamino-butyric acid (DAB)moiety. C-terminal modifications may further include replacing the freeacid with a carboxamide group or forming a cyclic lactam at the carboxyterminus to introduce structural constraints.

Naturally occurring side chains of the 20 genetically encoded aminoacids (or D amino acids) may be replaced with other side chains withsimilar properties, for instance with groups such as alkyl, lower alkyl,cyclic 4-, 5-, 6-, to 7-membered alkyl, amide, amide lower alkyl, amidedi(lower alkyl), lower alkoxy, hydroxy, carboxy and the lower esterderivatives thereof, and with 4-, 5-, 6-, to 7-membered heterocyclic.

Such substitutions can include but are not necessarily limited to: (1)non-standard positively charged amino acids, like: ornithine;N-(4-aminobutyl)-glycine having a lysine side chain attached to the“N-terminus” and aminopropyl or aminoethyl groups attached to the aminogroup of glycine; (2) Non-naturally occurring amino acids with no netcharge and sidechains similar to arginine, such as citrulline, with orwithout methylene groups; (3) non-standard non-naturally occurring aminoacids with OH (e.g., serine), such as, homoserine, hydroxyproline,hydroxyvaline, and penicillamin; (4) proline derivatives, such as,D-Pro, including 3,4-dehydroproline, pyroglutamine, proline withfluorine substitutions on the ring, 1,3-thiazolidine-4-carboxylic acid;(5) Histidine derivative, such as beta-(2-thienyl)-alanine; or (6) alkylderivatives, such as 2-aminobutyric acid, norvaline, norleucine,homoleucine, and alpha-aminoisobutyric acid.

In other embodiments, the C-terminal carboxyl group or a C-terminalester may be induced to cyclize by internal displacement of the —OH orthe ester (—OR) of the carboxyl group or ester respectively with theN-terminal amino group to form a cyclic peptide. For example, aftersynthesis and cleavage to give the peptide acid, the free acid isconverted to an activated ester by an appropriate carboxyl groupactivator such as dicyclohexylcarbodiimide (DCC) in solution, forexample, in methylene chloride (CH₂Cl₂), dimethyl formamide (DMF)mixtures. The cyclic peptide is then formed by internal displacement ofthe activated ester with the N-terminal amine. Internal cyclization asopposed to polymerization can be enhanced by use of very dilutesolutions. Such methods are well known in the art.

In other embodiments, the multipartite peptide of the present disclosureis cyclized or includes a desamino or descarboxy residue at the peptidetermini so that there are no terminal amino or carboxyl groups. This candecrease susceptibility to proteases and/or to restrict the conformationof the peptide. C-terminal functional groups of the compounds of thepresent disclosure include amide, amide lower alkyl, amide di(loweralkyl), lower alkoxy, hydroxy, and carboxy, and the lower esterderivatives thereof, and the pharmaceutically acceptable salts thereof.The multipartite peptide may be cyclized by adding an N and/or Cterminal cysteine and cyclizing the peptide through disulfide linkagesor other side chain interactions.

In one preferred embodiment, the multipartite peptide (or pharmaceuticalcomposition thereof) has the structure A-B-C-D, where A is a PEG, suchas a 10 kD PEG; B is a peptide cleavage linker sequence for anendopeptidase, such as asparagine endopeptidase; C is an SMR-containingpeptide sequence, such as VGFPVAAVGFPV (SEQ ID NO:2); and D is a Clu-BPpeptide sequence, such as HPLSKHPYWSQP (SEQ ID NO:3).

The multipartite peptides of the present disclosure may be administeredas naked peptides with or without PEG moieties, or they may beincorporated into suitable carriers, such as liposomes, nanoparticles,hydrogels, microcapsules, viruses or bacteriophages, or virus-likeparticles (VLPs).

Peptide-Encoding Polynucleotides

Another aspect of the present disclosure relates to a polynucleotideencoding any of the multipartite peptides described herein. In oneembodiment, the polynucleotide is an expression vector. As used herein,the term “expression vector” refers to a non-viral or a viral vectorthat comprises a polynucleotide encoding the multipartite peptide of thepresent disclosure in which the peptide coding sequences are operablylinked to regulatory sequences sufficient for expressing the peptide ina cell. One type of non-viral vector is a “plasmid”, which includes acircular double-stranded DNA loop into which additional DNA segments canbe ligated. In the present specification, “plasmid” and “vector” can beused interchangeably as the plasmid is the most commonly used form ofvector.

The regulatory sequences may be selected on the basis of the host cellsto be used for expression, such that the design of the expression vectorand inclusion of regulatory sequences depends on such factors as thechoice of the host cell to be transformed, the level of expression ofprotein desired, whether the peptide is to be secreted into theextracellular milieu and the like. The expression vectors of theinvention can be introduced into host cells to direct the expression ofthe multipartite peptide of the present disclosure in vitro forproduction purposes or in vivo for therapeutic purposes.

As used herein, the terms “control sequences” or “regulatory sequences”refer to DNA sequences necessary for the expression of an operablylinked coding sequence in a particular host organism. The term“control/regulatory sequence” is intended to include promoters,enhancers and other expression control elements (e.g., polyadenylationsignals). Control/regulatory sequences include those which directconstitutive expression of a nucleotide sequence in many types of hostcells and those which direct expression of the nucleotide sequence onlyin certain host cells (e.g., tissue-specific regulatory sequences).

A nucleic acid sequence is “operably linked” to another nucleic acidsequence when the former is placed into a functional relationship withthe latter. For example, in certain embodiments, the expression vectorencodes a presequence or signal peptide that is operably linked to thepeptide coding sequences for expression as a preprotein thatparticipates in the secretion of the polypeptide. In addition, apromoter or enhancer is said to be operably linked to a coding sequenceif it affects the transcription of the sequence and a ribosome bindingsite is operably linked to a coding sequence if it is positioned so asto facilitate translation. Generally, “operably linked” means that theDNA sequences being linked are contiguous and, in the case of asecretory leader, contiguous and in reading phase. However, enhancers donot have to be contiguous. Linking these sequences may be accomplishedby ligation at convenient restriction sites or by the use of syntheticoligonucleotide adaptors, primers, and/or linkers are used in accordancewith conventional practices in the art.

In certain cases, these vectors may be engineered to target certaindiseases or cell populations by using the targeting characteristicsinherent to the virus vector or engineered into the virus vector.Specific cells may be “targeted” for delivery of polynucleotides, aswell as expression. Thus, the term “targeting”, in this case, may bebased on the use of endogenous or heterologous binding agents in theform of capsids, envelope proteins, antibodies for delivery to specificcells, the use of tissue-specific regulatory elements for restrictingexpression to specific subset(s) of cells, or both.

In certain embodiments, the expression vector is engineered to directexpression of the peptide ubiquitously or preferentially in a particularcell type (e.g., tissue-specific regulatory elements are used to expressthe polynucleotide). Thus, in certain embodiments, expression of thepeptide is under the control of a tissue specific or ubiquitouspromoter, such as the CMV promoter or a CMV-chicken beta-actin hybrid(CAG) promoter. In other embodiments, a tissue specific ortumor-specific promoter may be used. Exemplary tissue-specificregulatory elements are known in the art and may include liver-specificpromoter (e.g., albumin promoter), lymphoid-specific promoters,epithelial cell-specific promoters, promoters of T cell receptors andimmunoglobulins, neuron-specific promoters (e.g., the neurofilamentpromoter), pancreas-specific promoters (e.g., insulin promoter), andmammary gland-specific promoters (e.g., milk whey promoter).Developmentally-regulated promoters (e.g., the α-fetoprotein promoter)are also encompassed.

In certain embodiments, the multipartite expression construct may belinked to a mitochondrial targeting peptide or leader sequence tofacilitate uptake of the expression construct into mitochondrial cellsas described in U.S. Patent Publication No. 20040192627. Conventionalprotocols may be used to conjugate the expression construct with themitochondrial targeting peptide, e.g., pGeneGrip™ technology(Genlantis/Gene Therapy Systems, Inc., San Diego, Calif.).Alternatively, the multipartite peptide coding sequence may be fused toa mitochondrial targeting sequence to direct translocation of theexpressed peptide into mitochondria as described above.

In other embodiments, the multipartite peptide coding sequence may befused to a mitochondrial penetrating moiety or mitochondrial targetingsignal sequence as described above. Exemplary nucleic acids that act asmitochondrial penetrating moieties (such as those described in U.S. Pat.No. 5,569,754) include e.g., CCGCCAAGAAGCG (SEQ ID NO:31),GCGTGCACACGCGCGTAGACTTCCCCCGCAAGTCACTCGTTAGCCCGCCAAGAAGCGACCCCTCCGGGGCGAGCTGAGCGGCGTGGCGCGGGGGCGTCAT (SEQ ID NO:32),ACGTGCATACGCACGTAGACATTCCCCGCTTCCCACTCCAAAGTCCGCCAAGAAGCGTATCCCGCTGAGCGGCGTGGCGCGGGGGCGTCATCCGTCAGCTC (SEQ ID NO:33) orACTTCCCCCGCAAGTCACTCGTTAGCCCGCCAAGAAGCGACCCCTCCGGGGCGAGCT G (SEQ IDNO:34).

In some embodiments, the multipartite peptide coding sequence may befused to a signal peptide domain for secretion of the peptide from cellsexpressing the peptide. The signal peptide sequence is removed from themature peptide as the mature peptide is secreted from the cell. Since agiven signal peptide sequence can affect the level of peptideexpression, a peptide-encoded polynucleotide may include any one of avariety of different N-terminal signal peptide sequences known in theart.

In some embodiments, the expression vector is a viral vector. A viralvectors may be derived from an adeno-associated virus (AAV), adenovirus,herpesvirus, vaccinia virus, poliovirus, poxvirus, a retrovirus(including a lentivirus, such as HIV-1 and HIV-2), Sindbis and other RNAviruses, alphavirus, astrovirus, coronavirus, orthomyxovirus,papovavirus, paramyxovirus, parvovirus, picornavirus, togaviruses andthe like. A non-viral vector is simply a “naked” expression vector thatis not packaged with virally derived components (e.g., capsids and/orenvelopes).

Non-viral expression vectors can be utilized for non-viral genetransfer, either by direct injection of naked DNA or by encapsulatingthe multipartite peptide-encoding polynucleotides in liposomes,nanoparticles, hydrogels, microcapsules, or virus-like particles. Suchcompositions can be further linked by chemical conjugation to targetingdomains to facilitate targeted delivery and/or entry of nucleic acidsinto desired cells of interest. In addition, plasmid vectors may beincubated with synthetic gene transfer molecules such as polymericDNA-binding cations like polylysine, protamine, and albumin, and linkedto cell targeting ligands such as asialoorosomucoid, insulin, galactose,lactose or transferrin.

Alternatively, naked DNA may be employed. Uptake efficiency of naked DNAmay be improved by compaction or by using biodegradable latex beads.Such delivery may be improved further by treating the beads to increasehydrophobicity and thereby facilitate disruption of the endosome andrelease of the DNA into the cytoplasm.

Pharmaceutical Compositions

In some embodiments, a pharmaceutical composition comprises amultipartite peptide comprising at least one SMR peptide from HIV-1 Nef,at least one Clusterin (Clu)-binding peptide (Clu-BP) and apharmaceutically acceptable carrier. In addition, the multipartitepeptide may include any of the above described modification.

In another embodiment, a pharmaceutical composition comprises anexpression vector encoding a multipartite peptide comprising at leastone SMR peptide from HIV-1 Nef, at least one Clusterin (Clu)-bindingpeptide (Clu-BP) and a pharmaceutically acceptable carrier, whereby theencoded peptide is designed to include any of the above describedmodifications.

As used herein, the term “pharmaceutically acceptable” refers to amolecular entity or composition that does not produce an adverse,allergic or other untoward reaction when administered to an animal or ahuman, as appropriate. The term “pharmaceutically acceptable carrier”,as used herein, includes any and all solvents, solubilizers, fillers,stabilizers, surfactants, binders, absorbents, bases, buffering agents,excipients, lubricants, controlled release vehicles, diluents,emulsifying agents, humectants, lubricants, gels, dispersion media,coatings, antibacterial or antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration. The use of such carriers and agents for pharmaceuticallyactive substances is well-known in the art. See e.g., A. H. KibbeHandbook of Pharmaceutical Excipients, 3rd ed. Pharmaceutical Press,London, UK (2000).

Exemplary carriers or excipients include but are not limited to, calciumcarbonate, calcium phosphate, various sugars, starches, cellulosederivatives, gelatin, polymers such as polyethylene glycols, water,saline, isotonic aqueous solutions, phosphate buffered saline, dextrose,0.3% aqueous glycine, glycerol, ethanol and the like, as well ascombinations thereof. In many cases, it will be preferable to includeisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride in the composition, or glycoproteins forenhanced stability, such as albumin, lipoprotein and globulin.Pharmaceutically acceptable carriers may further comprise minor amountsof auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the therapeutic agents. In certain embodiments, the pharmaceuticallyacceptable carrier comprises serum albumin.

Formulation characteristics that can be modified include, for example,pH and osmolality. For example, it may be desired to achieve aformulation that has a pH and osmolality similar to that of human bloodor tissues to facilitate the formulation's effectiveness whenadministered parenterally.

Buffers are useful in the present invention for, among other purposes,manipulation of the total pH of the pharmaceutical formulation(especially desired for parenteral administration). A variety of buffersknown in the art can be used in the present formulations, such asvarious salts of organic or inorganic acids, bases, or amino acids, andincluding various forms of citrate, phosphate, tartrate, succinate,adipate, maleate, lactate, acetate, bicarbonate, or carbonate ions.Particularly advantageous buffers for use in parenterally administeredforms of the presently disclosed compositions in the present inventioninclude sodium or potassium buffers, including sodium phosphate,potassium phosphate, sodium succinate and sodium citrate.

Sodium chloride can be used to modify the tonicity of the solution at aconcentration of 0-300 mM (optimally 150 mM for a liquid dosage form).Cryoprotectants can be included for a lyophilized dosage form,principally 0-10% sucrose (optimally 0.5-1.0%). Other suitablecryoprotectants include trehalose and lactose. Bulking agents can beincluded for a lyophilized dosage form, principally 1-10% mannitol(optimally 2-4%). Stabilizers can be used in both liquid and lyophilizeddosage forms, principally 1-50 mM L-Methionine (optimally 5-10 mM).Other suitable bulking agents include glycine, arginine, can be includedas 0-0.05% polysorbate-80 (optimally 0.005-0.01%).

In one embodiment, sodium phosphate is employed in a concentrationapproximating 20 mM to achieve a pH of approximately 7.0. A particularlyeffective sodium phosphate buffering system comprises sodium phosphatemonobasic monohydrate and sodium phosphate dibasic heptahydrate. Whenthis combination of monobasic and dibasic sodium phosphate is used,advantageous concentrations of each are about 0.5 to about 1.5 mg/mlmonobasic and about 2.0 to about 4.0 mg/ml dibasic, with preferredconcentrations of about 0.9 mg/ml monobasic and about 3.4 mg/ml dibasicphosphate. The pH of the formulation changes according to the amount ofbuffer used.

Depending upon the dosage form and intended route of administration itmay alternatively be advantageous to use buffers in differentconcentrations or to use other additives to adjust the pH of thecomposition to encompass other ranges. Useful pH ranges for compositionsof the present invention include a pH of about 2.0 to a pH of about12.0.

In some embodiments, it will also be advantageous to employ surfactantsin the presently disclosed formulations, where those surfactants willnot be disruptive of the drug-delivery system used. Surfactants oranti-adsorbants that prove useful include polyoxyethylenesorbitans,polyoxyethylenesorbitan monolaurate, polysorbate-20, such as Tween-20™,polysorbate-80, polysorbate-20, hydroxycellulose, genapol and BRIJsurfactants. By way of example, when any surfactant is employed in thepresent invention to produce a parenterally administrable composition,it is advantageous to use it in a concentration of about 0.01 to about0.5 mg/ml.

Additional useful additives are readily determined by those of skill inthe art, according to particular needs or intended uses of thecompositions and formulator. One such particularly useful additionalsubstance is sodium chloride, which is useful for adjusting theosmolality of the formulations to achieve the desired resultingosmolality. Particularly preferred osmolalities for parenteraladministration of the disclosed compositions are in the range of about270 to about 330 mOsm/kg. The optimal osmolality for parenterallyadministered compositions, particularly injectables, is approximately300 mOsm/kg and achievable by the use of sodium chloride inconcentrations of about 6.5 to about 7.5 mg/ml with a sodium chlorideconcentration of about 7.0 mg/ml being particularly effective.

Multipartite peptides can be stored as a lyophilized powder underaseptic conditions and combined with a sterile aqueous solution prior toadministration. The aqueous solution used to resuspend the peptides cancontain pharmaceutically acceptable auxiliary substances as required toapproximate physical conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents and the like, as discussed above.Alternatively, the multipartite peptides can be stored as a suspension,preferable an aqueous suspension, prior to administration.

In certain preferred embodiments, the multipartite peptide in thepharmaceutical composition is pegylated. PEGylation is a process forcovalently attaching polyethylene glycol polymer chains to anothermolecule, normally a drug or therapeutic peptide/protein. PEGylation canbe achieved by incubation of a reactive derivative of PEG with themultipartite peptide. The covalent attachment of PEG to a multipartitepeptide can “mask” the multipartite peptide from the host's immunesystem (reduced immunogenicity and antigenicity), increase thehydrodynamic size (size in solution) of the multipartite peptide whichprolongs its circulatory time by reducing renal clearance. PEGylationcan also provide water solubility to hydrophobic proteins.

The PEG molecules are typically characterized as having for example fromabout 2 to about 1000, or from about 2 to about 300 repeating units. Forexample water-soluble polymers, including but not limited to PEG,poly(ethylene oxide) (PEO), polyoxyethylene (POE), polyvinyl alcohols,hydroxyethyl celluloses, or dextrans, are commonly conjugated toproteins to increase stability or size, etc., of the protein asdescribed in U.S. Patent Publication No. 2012/0171115.

PEG, PEO and POE are oligomers or polymers of ethylene oxide. In thecase of PEG, these oligomers or polymers are produced by, e.g., anionicring opening polymerization of ethylene oxide initiated by nucleophilicattack of a hydroxide ion on the epoxide ring. One of the more usefulforms of PEG for protein modification is monomethoxy PEG (mPEG).

Preferred PEGs are monodisperse or polydisperse, preferablymonodisperse. The skilled artisan will be aware that PEG can bepolydisperse or monodisperse. Polydisperse PEG comprises a mixture ofPEGs having different molecular weights. In the case of polydispersePEGs, reference to a specific molecular weight will be understood torefer to the number average molecular weight of PEGs in the mixture. Thesize distribution is characterized statistically by its weight averagemolecular weight (MW) and its number average molecular weight (Mn), theratio of which is called the polydispersity index (Mw/Mn). MW and Mn aremeasured, in certain aspects, by mass spectroscopy. Most of thePEG-protein conjugates, particularly those conjugated to PEG larger than1 KD, exhibit a range of molecular weights due to a 30 polydispersenature of the parent PEG molecule. For example, in case of mPEG2K(Sunbright ME-020HS, NOF), actual molecular masses are distributed overa range of 1.5-3.0 KD with a polydispersity index of 1.036. Based on theforegoing, the skilled artisan will be aware that monodisperse PEGcomprises a mixture of PEGs comprising substantially the same molecularweight. Monodisperse PEGs are commercially available, e.g., fromPolypure AS, Norway.

The average or preferred molecular weight of the PEG may range from 500Da to 200 kDa, from 1 to 100 kDa, from 2 to 50 kDa, from 5 to 25 kDa, orfrom 5 kDa to 10 kDa, including any integers encompassed within theseranges.

The choice of the suitable functional group for the PEG derivative isbased on the type of available reactive group on the molecule that willbe coupled to the PEG. For peptides or proteins, typical reactive aminoacids include lysine, cysteine, histidine, arginine, aspartic acid,glutamic acid, serine, threonine, tyrosine. The N-terminal amino groupand the C-terminal carboxylic acid can also be used as a site specificsite by conjugation with aldehyde functional polymers.

In certain embodiments, the PEG derivatives are produced by reacting thePEG polymer with a group that is reactive with hydroxyl groups,typically anhydrides, acid chlorides, chloroformates and carbonates. Inother embodiments, more efficient functional groups such as aldehyde,esters, amides, etc. are made available for protein conjugation.

In certain embodiments, heterobifunctional PEGs are used forconjugation. These heterobifunctional PEGs are useful for linking twoentities, where a hydrophilic, flexible and biocompatible spacer isneeded. Preferred end groups for heterobifunctional PEGs are maleimide,vinyl sulfones, pyridyl disulfide, amine, carboxylic acids and NHSesters. In other embodiments, the pegylation agents contain branched, Yshaped or comb shaped polymers that show reduced viscosity and lack oforgan accumulation.

Various methods are known in the art for conjugating PEGs to peptides orproteins, as describe in U.S. Patent Publication No. 2012/0171115.Conjugation of PEGs may include the use of spacer moieties that arecleavable or non-cleavable. In some embodiments, the cleavable spacermoiety is a redox-cleavable spacer moiety, such that the spacer moietyis cleavable in environments with a lower redox potential, such thecytoplasm and other regions with higher concentrations of molecules withfree sulfhydryl groups. Examples of spacer moieties that may be cleaveddue to a change in redox potential include those containing disulfides.The cleaving stimulus can be provided upon intracellular uptake of theconjugated protein where the lower redox potential of the cytoplasmfacilitates cleavage of the spacer moiety. In the case of PEG, themolecule can be activated to facilitate its binding to amines orimidazoles, a carboxylic group, a hydroxyl group or a sulfhydryl group.

In another example, a decrease in pH causes cleavage of the spacer tothereby release of the compound into a target cell. A decrease in pH isimplicated in many physiological and pathological processes, such asendosome trafficking, tumour growth, inflammation, and myocardialischemia. The pH drops from a physiological 7.4 to 5-6 in endosomes or4-5 in lysosomes. Examples of acid sensitive spacer moieties which maybe used to target lysosomes or endosomes of cancer cells, include thosewith acid-cleavable bonds such as those found in acetals, ketals,orthoesters, hydrazones, trityls, cis-aconityls, or thiocarbamoyls (seefor example, U.S. Pat. Nos. 4,569,789, 4,631,190, 5,306,809, and5,665,358). Other exemplary acid-sensitive spacer moieties comprisedipeptide sequences Phe-Lys and Val-Lys.

Cleavable spacer moieties may be sensitive to biologically suppliedcleaving agents that are associated with a particular target cell, forexample, lysosomal or tumor-associated enzymes. Examples of linkingmoieties that can be cleaved enzymatically include, but are not limitedto, esters and endopeptidase cleavage recognition sites.

An activated PEG may be used with cyanuric chloride to produce a PEGdichlorotriazine derivative. This derivative can react with multiplefunctional nucleophilic functional groups, such as lysine, serine,tyrosine, cysteine and histidine. Two widely used forms of PEG used toconjugate to proteins are succinimidyl carbonate PEG and benzotriazolecarbonate PEG (BTC-PEG; U.S. Pat. No. 5,560,234). Both of thesecompounds react preferentially with lysine residues to form carbamatelinkages, however are also known to react with hystidine and tyrosine.SC-PEG is slightly more resistant to hydrolysis than BTC-PEG.

Another PEG useful for conjugating to proteins is PEG-propionaldehyde(U.S. Pat. No. 5,252,714). An advantage of this chemistry is that underacidic conditions (about pH5) it is largely selective for N-terminalα-amine thus avoiding potential problems with non-specific conjugation.An acetal derivative of PEG-propionaldehyde, i.e., PEG-acetalaldehydeprovides an additional benefit in so far as it provides for longerstorage than PEG-propionaldehyde (U.S. Pat. No. 5,990,237).

Active esters of PEG carboxylic acids are probably one of the most usedacylating agents for protein conjugation. Active esters react withprimary amines near physiological conditions to form stable amides.Activation of PEG-carboxylic acids to succinimidyl active esters isaccomplished by reacting the PEG-carboxylic acid withN-hydroxysuccinimide (NHS or HOSu) and a carbodiimide. Exemplarycarboxylic acid derivatives of PEG include carboxymethylated PEG(CM-PEG), butanoic acid derivatives and propionic acid derivatives (U.S.Pat. No. 5,672,662). Changing the distance between the active ester andthe PEG backbone by the addition of methylene units can dramaticallyinfluence reactivity towards water and amines (e.g., by reducinghydrolysis). Alternatively or in addition, hydrolysis can be reduced byintroducing an .alpha.-branching moiety to the carboxylic acid.

PEGylation of free cysteine residues in a protein is useful forsite-specific conjugation (e.g., using a protein modified to includecysteine residues as described herein). Exemplary PEG derivatives forcysteine conjugation include PEG-maleimide, PEG-vinylsulfone,PEG-iodoacetamide and PEG-orthopyridyl disulfide. Exemplary methods forconjugating PEG to cysteine residues and for conjugation usingPEG-vinylsulfone are well known in the art.

U.S. Pat. No. 5,985,263 describes methods for conjugating PEG to thesecondary amine group of histidine, which has a lower pKa than theprimary amine. An advantage of this approach is that the acyl-histidinebond is not stable meaning that the peptide or protein is slowlyreleased (i.e., the conjugate behaves as a slow release formulation or apro-drug).

Another approach for PEGylation is to take advantage of a N-terminalserine or threonine, which can be converted to periodate as discussedabove. Using this approach, PEG has been conjugated to bioactiveproteins (e.g., Gaertner and Offord, 1996). PEG can also be conjugatedto carbohydrate groups.

The pharmaceutical composition of the present disclosure is formulatedto be compatible with its intended route of administration. Examples ofroutes of administration include parenteral, e.g., intrathecal,intra-arterial, intravenous, intradermal, subcutaneous, oral,transdermal (topical) and transmucosal administration. Solutions orsuspensions used for parenteral, intradermal, or subcutaneousapplication can include the following components: a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine; propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfate; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. pH can be adjusted with acids or bases,such as hydrochloric acid or sodium hydroxide. The parenteralpreparation can be enclosed in ampoules, disposable syringes or multipledose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CREMOPHOREL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the injectable composition should be sterile and should be fluidto the extent that easy syringability exists. It must be stable underthe conditions of manufacture and storage and must be preserved againstthe contaminating action of microorganisms such as bacteria and fungi.The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyetheylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequited particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, and sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating themultipartite peptide in the required amount in an appropriate solventwith one or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the multipartite peptide into a sterile vehicle whichcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze-drying which yields a powder ofthe active peptide plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. The tablets, pills, capsules, troches and the like can containany of the following ingredients, or compounds of a similar nature,including a binder such as microcrystalline cellulose, gum tragacanth orgelatin; an excipient such as starch or lactose, a disintegrating agentsuch as alginic acid, Primogel, or corn starch; a lubricant such asmagnesium stearate or Stertes; a glidant such as colloidal silicondioxide; a sweetening agent such as sucrose or saccharin; or a flavoringagent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the peptides are delivered in the formof an aerosol spray from pressured container or dispenser which containsa suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the pharmaceutical compositions areformulated into ointments, salves, gels, or creams as generally known inthe art.

In certain embodiments, the pharmaceutical composition is formulated forcontrolled or delayed release of the active ingredient. For example, incertain embodiments, the peptides may be delivered from with an entericcoating applied as a barrier to an oral formulation so as to preventrelease of the peptides before they reach the small intestine. As usedherein, the term “enteric coating” is a coating comprising of one ormore polymers having a pH dependent or pH-independent release profile.An enteric coated pill will not dissolve in the acidic juices of thestomach (pH˜3), but they will in the alkaline (pH 7-9) environmentpresent in the small intestine or colon. An enteric polymer coatingtypically resists releases of the active agents until sometime after agastric emptying lag period of about 3-4 hours after administration.

Such enteric coatings or barrier coatings are also used to protectacid-unstable peptides from the stomach's acidic exposure, deliveringthem instead to a basic pH environment (intestine's pH 5.5 and above)where they do not degrade and can mediate their desired action. An oralformulation may comprise a plurality of barrier coatings comprising avariety of different materials to facilitate release in a temporalmanner. The coating may be a sugar coating, a film coating (e.g., basedon hydroxypropyl methylcellulose, methylcellulose, methylhydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose,acrylate copolymers, polyethylene glycols, and/or polyvinylpyrrolidone)or a coating based on methacrylic acid copolymer, cellulose acetatephthalate, hydroxypropyl methylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, polyvinyl acetate phthalate, shellac,and/or ethylcellulose. Furthermore, the formulation may additionallyinclude a time delay material such as glyceryl monostearate or glyceryldistearate.

Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. The materialscan also be obtained commercially from e.g. Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to infected cells with monoclonal antibodies to tumor antigensor viral antigens) can also be used as pharmaceutically acceptablecarriers.

It is especially advantageous to formulate the peptide compositions indosage unit form for ease of administration and uniformity of dosage.Suitable unit dosage forms include, but are not limited to powders,tablets, pills, capsules, lozenges, suppositories, patches, nasalsprays, injectibles, implantable sustained-release formulations, lipidcomplexes, etc.

Methods of Treatment

In another aspect, the present disclosure provides methods for treatingcancers and infectious diseases. In some embodiments, the presentapplication relates to a method for treating cancer. The methodcomprises the step of administering to a subject in need thereof aneffective amount of a pharmaceutical composition comprising amultipartite peptide containing at least one secretion modifying region(SMR) peptide from HIV-1 Nef and at least one Clusterin (Clu)-bindingpeptide (Clu-BP). The subject may have a cancer selected from the groupconsisting of leukemia, lymphoma, carcinoma, melanoma, sarcoma, germcell tumor and blastoma.

As used herein, the term “leukemia” refers to progressive, malignantdiseases of the blood-forming organs and is generally characterized by adistorted proliferation and development of leukocytes and theirprecursors in the blood and bone marrow. Exemplary leukemias include,for example, acute nonlymphocytic leukemia, chronic lymphocyticleukemia, acute granulocytic leukemia, chronic granulocytic leukemia,acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia,a leukocythemic leukemia, basophylic leukemia, blast cell leukemia,bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonalleukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia,hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia,stem cell leukemia, acute monocytic leukemia, leukopenic leukemia,lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia,lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia,mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia,monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloidgranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasmacell leukemia, plasmacytic leukemia, promyelocytic leukemia, Rieder cellleukemia, Schilling's leukemia, stem cell leukemia, subleukemicleukemia, and undifferentiated cell leukemia.

The term “lymphoma” refers to a group of blood cell tumors that developfrom lymphocytes. Exemplary lymphomas include, for example, Hodgkin'slymphomas (HL) and the non-Hodgkin lymphomas.

The term “carcinoma” refers to the malignant growth of epithelial cellstending to infiltrate the surrounding tissues and give rise tometastases. Exemplary carcinomas include, for example, acinar carcinoma,acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma,carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma,alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare,basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolarcarcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriformcarcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloidcarcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma,carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma,cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonalcarcinoma, encephaloid carcinoma, epiennoid carcinoma, carcinomaepitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere,carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giantcell carcinoma, carcinoma gigantocellulare, glandular carcinoma,granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma,hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma,hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma insitu, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher'scarcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticularcarcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelialcarcinoma, carcinoma medullare, medullary carcinoma, melanoticcarcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum,carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum,mucous carcinoma, carcinoma myxomatodes, naspharyngeal carcinoma, oatcell carcinoma, carcinoma ossificans, osteoid carcinoma, papillarycarcinoma, periportal carcinoma, preinvasive carcinoma, prickle cellcarcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reservecell carcinoma, carcinoma sarcomatodes, schneiderian carcinoma,scirrhous carcinoma, carcinoma scroti, signet-ring cell carcinoma,carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidalcell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamouscarcinoma, squamous cell carcinoma, string carcinoma, carcinomatelangiectaticum, carcinoma telangiectodes, transitional cell carcinoma,carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, andcarcinoma villosum.

The term “sarcoma” refers to a tumor made up of a substance like theembryonic connective tissue and is generally composed of closely packedcells embedded in a fibrillar or homogeneous substance. Exemplarysarcomas include, for example, chondrosarcoma, fibrosarcoma,lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy'ssarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma,ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, choriocarcinoma, embryonal sarcoma, Wilns' tumor sarcoma, endometrial sarcoma,stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma,giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathicmultiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of Bcells, lymphomas (e.g., Non-Hodgkin Lymphoma), immunoblastic sarcoma ofT-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma,angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parostealsarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma,synovial sarcoma, and telangiectaltic sarcoma.

The term “melanoma” refers to a tumor arising from the melanocyticsystem of the skin and other organs. Melanomas include, for example,acral-lentiginous melanoma, amelanotic melanoma, benign juvenilemelanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma,juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodularmelanoma subungal melanoma, and superficial spreading melanoma.

Additional cancers include, for example, Hodgkin's Disease, multiplemyeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer,rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia,small-cell lung tumors, primary brain tumors, stomach cancer, coloncancer, malignant pancreatic insulanoma, malignant carcinoid,premalignant skin lesions, testicular cancer, thyroid cancer,neuroblastoma, esophageal cancer, genitourinary tract cancer, malignanthypercalcemia, cervical cancer, endometrial cancer, and adrenal corticalcancer. In one embodiment, the subject has breast cancer.

In other embodiments, the present application relates to a method fortreating an infectious disease. The method comprises the step ofadministering to a subject in need thereof an effective amount of apharmaceutical composition comprising a multipartite peptide containingat least one secretion modifying region (SMR) peptide from HIV-1 Nef andat least one Clusterin (Clu)-binding peptide (Clu-BP).

In some embodiments, the infectious disease is caused by a bacterium. Insome embodiments, the infectious disease is caused by a fungus, In someembodiments, the infectious disease is caused by a parasite.

In some embodiments, the infectious disease is caused by a virusselected from the group consisting of human immunodeficiency virus type1 and type 2 (HIV-1 and HIV-2), human T-cell lymphotropic virus type Iand type II (HTLV-I and HTLV-II), hepatitis A virus, hepatitis B virus(HBV), hepatitis C virus (HCV), hepatitis delta virus (HDV), hepatitis Evirus (HEV), hepatitis G virus (HGV), parvovirus B19 virus, hepatitis Avirus, hepatitis G virus, hepatitis E virus, transfusion transmittedvirus (TTV), Epstein-Barr virus, human cytomegalovirus type 1 (HCMV-1),human herpesvirus type 6 (HHV-6), human herpesvirus type 7 (HHV-7),human herpesvirus type 8 (HHV-8), influenza type A viruses, includingsubtypes H1N1 and H5N1, human metapneumovirus, severe acute respiratorysyndrome (SARS) coronavirus, hantavirus, and RNA viruses fromArenaviridae (e.g., Lassa fever virus (LFV)), Pneumoviridae (e.g., humanmetapneumovirus), Filoviridae (e.g., Ebola virus (EBOV), Marburg virus(MBGV) and Zika virus); Bunyaviridae (e.g., Rift Valley fever virus(RVFV), Crimean-Congo hemorrhagic fever virus (CCHFV), and hantavirus);Flaviviridae (West Nile virus (WNV), Dengue fever virus (DENV), yellowfever virus (YFV), GB virus C (GBV-C; formerly known as hepatitis Gvirus (HGV)); Rotaviridae (e.g., rotavirus), and combinations thereof.In one embodiment, the subject is infected with HIV-1 or HIV-2.

Dosages and Routes of Administration

Depending on the nature of the disease target, the multipartite peptidesof the present disclosure may be administered by any route, includingbut not limited to any of the various parenteral, gastrointestinal,inhalation, and topical (epicutaneous) routes of administration.Parenteral administration generally involves injections or infusions andincludes, for example, intravenous, intraarterial, intratumoral,intracardiac, intramuscular, intravesicular (e.g., to the bladder),intracerebral, intracerebroventricular, intraosseous infusion,intravitreal, intaarticular, intrathecal, epidural, intradermal,subcutaneous, transdermal, and intraperitoneal administration.Gastrointestinal administration includes oral, buccal, sublingual andrectal administration. The route of administration may involve local orsystemic delivery of the multipartite peptides.

As a general proposition, the therapeutically effective amount of themultipartite peptide administered will be in the range of about 1 ng/kgbody weight/day to about 100 mg/kg body weight/day whether by one ormore administrations. In a particular embodiment, the range ofmultipartite peptide administered is from about 1 ng/kg body weight/dayto about 1 μg/kg body weight/day, 1 ng/kg body weight/day to about 100ng/kg body weight/day, 1 ng/kg body weight/day to about 10 ng/kg bodyweight/day, 10 ng/kg body weight/day to about 1 μg/kg body weight/day,10 ng/kg body weight/day to about 100 ng/kg body weight/day, 100 ng/kgbody weight/day to about 1 μg/kg body weight/day, 100 ng/kg bodyweight/day to about 10 μg/kg body weight/day, 1 μg/kg body weight/day toabout 10 μg/kg body weight/day, 1 μg/kg body weight/day to about 100μg/kg body weight/day, 10 μg/kg body weight/day to about 100 μg/kg bodyweight/day, 10 μg/kg body weight/day to about 1 mg/kg body weight/day,100 μg/kg body weight/day to about 10 mg/kg body weight/day, 1 mg/kgbody weight/day to about 100 mg/kg body weight/day and 10 mg/kg bodyweight/day to about 100 mg/kg body weight/day.

In other embodiments, the multipartite peptide is administered at adosage range of 1 ng-10 ng per injection, 10 ng-100 ng per injection,100 ng-1 μg per injection, 1 μg-10 μg per injection, 10 μg-100 μg perinjection, 100 μg-1 mg per injection, 1 mg-10 mg per injection, 10mg-100 mg per injection, and 100 mg-1000 mg per injection. Themultipartite peptide may be injected daily, or every 2, 3, 4, 5, 6 and 7days.

In other embodiments, the dose range of the multipartite peptideadministered is from about 1 ng/kg to about 100 mg/kg. In still anotherparticular embodiment, the range of antibody administered is from about1 ng/kg to about 10 ng/kg, about 10 ng/kg to about 100 ng/kg, about 100ng/kg to about 1 μg/kg, about 1 μg/kg to about 10 μg/kg, about 10 μg/kgto about 100 μg/kg, about 100 μg/kg to about 1 mg/kg, about 1 mg/kg toabout 10 mg/kg, about 10 mg/kg to about 100 mg/kg, about 0.5 mg/kg toabout 30 mg/kg, and about 1 mg/kg to about 15 mg/kg.

In other particular embodiments, the amount of multipartite peptideadministered is, or is about, 0.0006, 0.001, 0.003, 0.006, 0.01, 0.03,0.06, 0.1, 0.3, 0.6, 1, 3, 6, 10, 30, 60, 100, 300, 600 and 1000 mg/day.

The specific dose of multipartite peptide is determined by theparticular circumstances of the individual patient including the size,weight, age and sex of the patient, the nature and stage of the disease,the aggressiveness of the disease, and the route of administration ofthe pharmaceutical composition.

In certain embodiments, the multipartite peptide may be administered atleast once per day, typically once, twice, three times or four times perday with the doses given at equal intervals throughout the day and nightin order to maintain a constant presence of the drug in order to providesufficient efficacy. However, a skilled artisan will appreciate that atreatment schedule can be optimized for any given patient, and thatadministration of compound may occur less frequently than once per day.

Dosage unit form as used herein includes physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of the multipartite peptidecalculated to produce the desired therapeutic effect in association withthe required pharmaceutical carrier. The specification for the dosageunit forms of the invention are dictated by and directly dependent onthe unique characteristics of the multipartite peptide and theparticular therapeutic effect to be achieved.

Toxicity and therapeutic efficacy of the multipartite peptide of thepresent disclosure can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., fordetermining the LD50 (the dose lethal to 50% of the population) and theED50 (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex and it can be expressed as the ratio LD50/ED50. Peptidesexhibiting large therapeutic indices are preferred. While peptides thatexhibit toxic side effects may be used, care should be taken to design adelivery system that targets such peptides to the site of affectedtissue in order to minimize potential damage to non-diseased cells and,thereby, reduce side effects.

Data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch peptides lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any peptide usedin the methods of the present disclosure, the therapeutically effectivedose can be estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. The pharmaceuticalcompositions can be included in a container, pack, or dispenser togetherwith instructions for administration.

When treating a cancer, any of the multipartite peptides of the presentdisclosure may be prescribed to be taken in combination with one or moreother anti-cancer agents. When used in such combination therapies, themultipartite peptides of the present disclosure and other pharmaceuticalagents may be administered simultaneously, by the same or differentroutes, or at different times during treatment. In particular, themultipartite peptides may be combined with a mortalin siRNA, ananti-cancer agent, such an alkylating agent; an anthracyclineantibiotic; an anti-metabolite; a detoxifying agent; an interferon; apolyclonal or monoclonal antibody; an EGFR inhibitor; a HER2 inhibitor;a histone deacetylase inhibitor; a hormone; a mitotic inhibitor; aphosphatidylinositol-3-kinase (PI3K) inhibitor; an Akt inhibitor; amammalian target of rapamycin (mTOR) inhibitor; a proteasomal inhibitor;a poly(ADP-ribose) polymerase (PARP) inhibitor; a Ras/MAPK pathwayinhibitor; a centrosome declustering agent; a multi-kinase inhibitor; aserine/threonine kinase inhibitor; a tyrosine kinase inhibitor; aVEGF/VEGFR inhibitor; a taxane or taxane derivative, an aromataseinhibitor, an anthracycline, a microtubule targeting drug, atopoisomerase poison drug, an inhibitor of a molecular target or enzyme(e.g., a kinase or a protein methyltransferase), a cytidine analogue,and combination thereof.

In some embodiments, the multipartite peptides of the present disclosureis administered in combination or concurrently with a chemotherapeuticagent, such as paclitaxel or cisplatin.

Likewise, when treating an infectious disease, the multipartite peptidesof the present disclosure may be prescribed to be taken in combinationwith one or more antiviral drugs. In certain embodiments, the antiviraldrug is a antiretroviral drug selected from the group consisting of:protease inhibitors, nucleoside reverse transcriptase inhibitors,nucleotide reverse transcriptase inhibitors, non-nucleoside reversetranscriptase inhibitors, integrase inhibitors, entry inhibitors, andmaturation inhibitors. Exemplary antiviral drugs include, but are notlimited to, abacavir, acyclovir, adefovir, amantadine, amdoxovir,amprenavir, antiprotease, apricitabine, arbidol, artemisinin,atazanafir, atripla, azidothymidine (AZT), bevirimat, boceprevir,butylated hydroxytoluene (BHT), cidofovir, combivir, darunavir,delavirdine, didanosine, dipivoxil, docosanol, edoxudine, efavirenz,elvitegravir, elvucitabine, emtricitabine, enfuviritide, entecavir,etravirine, famciclovir, foscarnet, fosamprenavir, gancyclovir,globoidnan A, GSK-572, HIV fusion inhibitors, hypericin, ibalizumab,idoxuridine, immunovir, indinavir, interferons (Types I, II and III),lamivudine, lersivirine, lopinivir, loviride, maraviroc, maribavir,MK-2048, molixan (NOV-205), moroxydine, nelfinavir, nevirapine, nexavir,non-nucleotide HIV RT inhibitors, oseltamivir, pegylated interferons(e.g., peginterferon alfa-2a), penciclovir, pencyclovir, peramivir,pleconaryl, podophyllotoxin, racivir, raltegravir, resquimod, ribavirin,rifampin, rilpivirine, rimantidine, ritonavir, saquinivir, stampidine,stavudine, taribavirin, tenofovir, tipranavir, trifluridine, trizivir,tromantidine, truvada, valaciclovir (Valtrex), valacyclovir,valganciclovir, vicriviroc, vidarabine, vivecon, zalcitabine, zanamivir(Relenza), zidovudine, and combinations thereof.

The treatment may be carried out for a period sufficient to achieve atherapeutic effect. Typically it is contemplated that treatment would becontinued indefinitely while the disease state persists, althoughdiscontinuation might be indicated if the pharmaceutical compositions nolonger produce a beneficial effect. The treating physician will know howto increase, decrease, or interrupt treatment based on patient response.

Production of the Multipartite Peptide

The multipartite peptides of the present disclosure can be chemicallysynthesized or produced from cells transformed with polynucleotideexpression vectors encoding the multipartite peptide. Multipartitepeptides of the present disclosure may be synthesized using traditionalliquid- or solid-phase synthesis. Fmoc and t-Boc solid phase peptidesynthesis (SPPS) can be employed to grow the peptides from carboxy toamino-terminus.

In other embodiments the multipartite peptides are synthesized usingrecombinant DNA technologies well known to those skilled in the art.Polynucleotide expression vectors can be designed to facilitatepreparative expression levels in many different cell hosts, includingbacteria, yeast, insect cells, and mammalian cells.

In one aspect, the present disclosure provides a host cell transformedwith a polynucleotide or expression vector encoding the multipartitepeptide. The host cells can be any bacterial or eukaryotic cell capableof expressing the multipartite peptide-encoding nucleic acids orexpression vectors described herein.

In another aspect, a method of producing a multipartite peptideaccording to the present disclosure comprises culturing a host celltransformed with a multipartite peptide-encoding polynucleotide orexpression vector under conditions that allows production of themultipartite peptide, and purifying the multipartite peptide from thecell. The peptides may be produced by culturing a cell transiently orstably expressing a multipartite peptide; and purifying the peptide fromthe cultured cells. Any cell capable of producing a functional peptidemay be used. The peptide-expressing cell may be of prokaryotic orbacterial origin, such as E. coli or it may be of eukaryotic ormammalian origin, such as a human cell. In other embodiments, the cellis a yeast cell or an insect cell. Where the cell is of eukaryoticorigin, the peptide-producing cell is preferably stably transformed witha polynucleotide so as to express the peptide.

The present disclosure is further illustrated by the following exampleswhich should not be construed as limiting. The contents of allreferences, patents and published patent applications cited throughoutthis application, as well as the Figures and Tables are incorporatedherein by reference.

Example 1: Materials and Methods

1-1. Cell Lines, Reagents and Antibodies.

The MCF-7 cell line, a noninvasive estrogen receptor positive (ER+) andMDA-MB-231 cell line (ER negative) were purchased from the American TypeCulture Collection (ATCC, Manassas, Va.). MCF-10A cell line, anon-tumorigenic epithelial cell line was also purchased from ATCC.3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT), Dulbecco'sModified Eagle's Medium (DMEM) with high glucose and FLUOROBRITE™ phenolred-free DMEM, (MCF-7) were purchased from Thermo Fisher Scientific(Rockford, Ill.) The RPMI 1640 medium (MDA-MB-231 cells) was obtainedfrom Life Technologies Company (Carlsbad, Calif.). The basal medium MEBMand the additive MEGM (MCF-10A cells) were obtained fromLonzal/Clonetics Corporation (Lonza, Walkersville, Md.). Paclitaxel waspurchased from Sellck-Chemon, (Houston, Tex.). Cisplatin was purchasedfrom EMD/Millipore (Billerica, Mass.). Annexin V-FITC/PI Apopto and PICell Cycle Kits were purchased from Nexcelom Bioscience (Lawrence,Mass.). The CD63 Rabbit polyclonal and Alix goat polyclonal antibodieswere purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, Calif.).The PEG-SMRwt-Clu PEG-SMRwt and PEG-SMRmut HIV-1 Nef peptides werepurchased from InnoPep Company (San Diego, Calif.).

1-2. Cell Culture.

Cells were cultured in the media described above with addition ofexosome-free fetal bovine serum (System Biosciences Inc., Mountain View,Calif.), 100 units/mL penicillin, and 100 mg/mL streptomycin andmaintained in a humidified atmosphere at 37° C. and 5% CO₂.

1-3. Viability and Proliferation.

Human breast cancer cell lines were seeded into 96-well plates (5000cells/well) and treated for 24 hours with various concentrations of SMRpeptides including PEG-SMRwt-Clu, PEG-SMRwt and PEG-SMRmut to determineIC50 (inhibition concentration). Cell proliferation was determined usingthe MTT assay (Molecular Devices, Sunnyvale, Calif.). Controlexperiments were performed with MTT treated cells alone and untreatedcells, and on this basis, the incubation times of 24 hr and 48 hr wereused for an MTT assay of peptide-treated cells (Stockerta J C. et al.,2012 and Riss T L. et al., 2015).

1-4. Cell Cycle Analysis.

MCF-7 and MDA-MB-231 breast cancer cells were cultured into 6-wellplates at 4×10⁵ cells per well and treated with either paclitaxel andcisplatin or combined with PEG-SMRwt-CLU peptide for 24 and 48 hours.Cell cycle analysis was performed using a propidium iodide cell cycleassay and measured using a Cellometer (Nexcelom, MA). Furtherexperiments were performed with SMR at IC50 concentration; the resultsshowed 1.12 μM of PEG-SMRwt-Clu, 0.28 μM of PEG-SMRwt on MCF-7 cells for24 hours and 0.28 μM of PEG-SMRwt-Clu, 0.42 μM of PEG-SMRwt onMDA-MB-231 cells for 24 hours for each cell time two stage. Breastcancer cells were seeded into 96-well plates at 5×10³ cells/ml, andtreated with either 1.6 μM/mL of paclitaxel, or 3 mg/mL cisplatin, 1.12PEG-SMRwt-CLU peptide, SMR peptide combined with paclitaxel or withcisplatin (MCF-7 cells). Alternatively 1.6 μM/mL paclitaxel or 2 mg/mLcisplatin or 0.28 μM/mL of PEG-SMR-CLU peptide, or the peptide combinedwith each of these drugs was used for MDA-MB-231 cells. Theconcentrations for cisplatin and paclitaxel were experimentallydetermined IC50 dosages for the different cell types (data not shown).At the end of the 24 hr or 48 hr incubations, the cells were assessed bythe Cellometry imaging cytometry assay.

All above steps were done on MCF-7 and MDA-MB-231 cells separately. Inorder to further understand whether this peptide functionssynergistically with chemotherapeutic drugs, 6 groups of cancer cellswere treated as follows: 1) untreated, 2) PEG-SMRwt-CLU, 3) paclitaxel,4) paclitaxel in combination with PEG-SMRwt-CLU, 5) cisplatin, 6)cisplatin in combination with PEG-SMRwt-CLU.

1-5. Assessment of Apoptosis.

Breast cancer cells were seeded into 6-well plates at 4×10⁵ cells perwell and treated with either paclitaxel or cisplatin or variousconcentrations of SMR peptides for 24 hours or different time point. SMRas described above. Apoptosis was determined the using AnnexinV-FITCdetection kit (Nexcelom, MA) and visualized by Cellometer imagingcytometry.

1-6. Exosome Isolation and Purification.

Exosomes were isolated from breast cancer cells by differentialcentrifugation as previously described (Ali S A. et al., 2010).Untreated tumor cells were used as a control. Briefly, the above treatedand untreated cell supernatants were centrifuged at 400×g for 10minutes. The supernatants were transferred to a clear tube andcentrifuged at 10,000×g for 30 minutes. The supernatants from the secondspin were ultracentrifuged at 200,000×g for 2 hours to pellet exosomes.Finally, the exosome pellets were re-suspended with PBS and stored at 4°C. until used for analysis.

1-7. Exosome Characterization by Acetylcholinesterase (AchE) Assay.

Purified exosomes were quantitated by measurement of AchE as described(Ellman et al., 1961). Briefly, a 100 mM dithibionitrobenzoic (DTNB)solution was prepared for use as a stock color indicator, and a 28.9mg/mL acetylthiocholine iodide in PBS solution was prepared as a stocksubstrate. The stock substrate stock can be stored at −20° C. up to onemonth, while the color indicator can be stored at 4° C. for two weeks. Aworking solution was prepared by mixing 10 mL of PBS with 200 μL ofSubstrate and 500 μl of DTNB. 50 μL of each exosome sample wastransferred to 96 well microtitre plates, and a standard curve wasprepared using AchE from 0.98 mU/mL to 2000 mU/mL. After 50 μL ofstandards were added into separate wells, 200 μL of the working solutionwas added to all wells. After 20 min incubation, AchE activity wasmeasured at 450 nm using a SpectroMax M5 fluorimeter.

1-8. Exosome Nanoparticle Tracking Analysis (NTA).

Analysis of absolute size distribution of exosomes was performed usingNanoSight LM10 with NTA2.3 (NanoSight Ltd., Minton Park, UK). Particleswere automatically tracked and sized based on Brownian motion and thediffusion coefficient. After isolation, the untreated and treated breastcancer exosomes were re-suspended in 0.5 mL of PBS. Control medium andfiltered PBS were used as controls in this technique. The NTAmeasurement conditions were: temperature=21.0+/−0.5° C.;viscosity=0.99+/−0.01 cP, frames per second=25, measurement time=30s.The detection threshold was similar in all samples. Two recordings wereperformed for each sample.

1-9. Western Blot Analysis.

Exosomes were isolated from culture supernatants as described above.Protein concentration was determined by measuring absorbance at 280 nm(Nanodrop 2000). Protein samples were denatured in SDS-PAGE samplebuffer by heating at 95° C. for 15 min. Criterion TGX Precast Gels(4-20% Bio-Rad, Richmond, Calif.) were used to separate the proteins andblotted as previously described (Huang M B. et al 2004). Blots wereincubated with the primary antibodies, anti-CD63 and anti-Alix, followedby goat or rabbit anti-Ig secondary antibodies. Specific bands weredetected using ECL chemiluminescent substrate (Santa Cruz Biotechnology,Santa Cruz, Calif.) and visualized on the ImageQuant LAS 4000 imagingsystem (GE Healthcare, Piscataway, N.J. 08854).

1-10. Fluorescent N-Rh-PE Measurement.

The fluorescent phospholipid analog N-Rh-PE [N-(lissamine rhodamine Bsulfonyl) phosphatidyl ethanolamine] is a lipid marker of exosomes andintraluminal vesicles of multivesicular bodies as previously described(Willem J et al., 1990). Briefly, 10 mM of the N-Rh-PE was stored inchloroform/methanol (2:1). A 5 μM N-Rh-PE solution in a pre-cooledreaction medium was then added to the treated with MCF-7 breast cancercells transfected with siRNA-Negative or siRNA-HSPA9, and then wereincubated at 4° C. for 1 h. After this incubation period, the medium wasremoved and the cells were extensively washed with cold medium to removeexcess unbound lipids. Labeled cells were cultured in complete RPMI-1640with 10% exosome-depleted FBS medium heat inactivated at 37° C.overnight. Measurement of N-Rh-PE in the collected supernatants/exosomeswas carried out using a spectrometer at 550 nm and 590 nm excitation andemission wavelengths, respectively.

1-11. Transfection with Mortalin Antibody.

MCF-7 breast cancer cells were transfected with mortalin antibody usinga Chariot kit (Active Motif, Carlsbad, Calif.) in accordance with themanufacturer's protocol. Following a 48 hour incubation of these cells,the exosomes were isolated and measured via AchE assay and NanoSightanalysis.

1-12. Transient Transfection with Small Interfering RNA (siRNA).

MCF-7 breast cancer cells were transfected with double-stranded siRNAsusing Amaxa's Nucleofector kit (Lonza Walkersville Inc., Walkersville,Md.) in accordance with the manufacturer's protocol. Transfection ofplasmids was carried out using Amaxa Biosystems Nucleofector II asrecommended by the supplier. Mortalin siRNAs were prepared as previouslydescribed (Shelton M N et al., 2012). Following transfection, the cellswere incubated at 37° C. for 24, 48, 72 and 96 hours, and exosomes wereisolated and measured by AchE assay and Western blotting.

1-13. Statistical Analysis.

Data was expressed as the mean±standard deviation (S.D.). A two-samplet-Test assuming equal variances was used to compare the differencesbetween controls and treated samples in each group. A value of p≤0.05was considered to be statistically significant.

Example 2: SMR Peptides Inhibit Cell Growth of Breast Cancer Cells

Breast cancer cells were treated for 24 hours with increasingconcentrations (35 nM/mL, 70 nM/mL, 140 nM/mL, 280 nM/mL, 560 nM/mL and1120 nM/mL) of PEG-SMRwt-Clu peptide in combination with eitherPEG-SMRwt or PEG-SMRmut peptides as controls. Both peptides containingthe SMRwt sequence inhibited breast cancer cell growth in adose-dependent manner (FIG. 1). For MCF-7 cells, 50% inhibition was seenwith 1.12 μM/mL of PEG-SMRwt-Clu and 0.28 μM/mL of PEG-SMRwt. ForMDA-MB-231 cells 50% inhibition was achieved with 0.28 μM/mL ofPEG-SMRwt-Clu and 0.42 μM/mL of PEG-SMRwt. The PEG-SMRmut peptide didnot inhibit proliferation.

Example 3: SMRwt Peptides Induce Cell Cycle Arrest in Breast CancerCells

The data indicated that PEG-SMRwt-CLU peptides induced cell cycle arrestin MCF-7 cells and MDA-MB-231 cells assayed at 48 hours (FIG. 2). Whencells were treated with the PEG-SMRwt-CLU peptide, or the peptidecombined with paclitaxel or cisplatin, they were blocked in G2/M phase,indicating that PEG-SMRwt-Clu peptides contribute to induction of G2/Marrest in breast cancer cells.

Example 4: SMRwt Peptides Increased the Sensitivity of Breast CancerCells to Cisplatin and Paclitaxel in MCF-7 Breast Cancer Cells

In a separate experiment, MCF-7 and MDA-MB-231 cells were treated witheither PEG-SMRwt-CLU or PEG-SMRmut-CLU alone, or in further combinationwith paclitaxel or cisplatin and then assayed for apoptosis by AnnexinV-FITC/PI assay. Both of these cell lines showed increased apoptosisrelative to the unmodified control peptides after the incubation withpaclitaxel and cisplatin for 48 hours (FIG. 3). Interestingly, thePEG-SMRwt-Clu peptide increased the level of drug-induced apoptosis inMCF-7 cells, but not in MDA-MB-231 cells.

Example 5: SMR Peptides Block Exosome Release in Breast Cancer Cells

Acetylcholinesterase (AchE) assays, NanoSight analysis and Western blotanalysis were performed to characterize exosomes released from MCF-7 andMDA-MB-231 human breast cancer cells treated for 48 hr with the variouspeptides. The results indicated that exosome release was inhibited bythe SMRwt peptides.

AchE activity in exosomes was assayed and the results of this analysisis shown in FIGS. 4A and 4B. In MCF-7 cells, the control exosomes werefound to contain 113.49 mU/mL of AchE activity. In contrast, 41.95 mU/mLof activity was found in cells treated with PEG-SMRwt-CLU peptide; 51.87mU/mL activity was found in cells treated with PEG-SMRwt-CLU incombination with paclitaxel; and 16.95 mU/mL activity was found in cellstreated with PEG-SMRwt-CLU in combination with (FIG. 4A). In MDA-MB-231cells, the control exosomes contained 118.48 mU/mL of AchE activity,whereas 66.77 mU/mL activity was found in cells treated withPEG-SMRwt-CLU peptide; 64.15 mU/mL activity was found in cells treatedwith PEG-SMRwt-CLU peptide in combination with paclitaxel; and 27.0mU/mL activity was found in cells treated with PEG-SMRwt-CLU incombination with cisplatin (FIG. 4B).

Analysis of exosomes concentration and size distribution was assayed byNanoSight LM10 Nanoparticle Tracking Analysis (NTA). With NTA, particlesare automatically tracked and sized based on Brownian motion and theassociated diffusion coefficient. Before analysis of the samples by NTA,it was determined that salt aggregates from the PBS did not contributeto background and the equipment was free of contaminant particles. Theuntreated MCF-7 cell control medium showed a considerable number ofparticles (5.16×10⁹ particles/ml) (FIG. 4 Panel C). However, a reducednumber of particles was found in MCF-7 cells treated with PEG-SMRwt-CLU(3.28×10⁸ particles/ml, p<2.40E-06), PEG-SMRwt-CLU in combination withpaclitaxel (5.7×10⁸ particles/mL, p<0.0008) and PEG-SMRwt-CLU incombination with cisplatin (3.77×10⁸ particles/mL, p<0.0001) (FIG. 4Panel C).

Similarly, whereas control media from MDA-MB-231 cultures also showed aconsiderable number of particles (4.7×10⁹ particles/ml), a reducednumber of particles was found in MDA-MB-231 cells treated withPEG-SMRwt-CLU peptide (6.8×10⁸ particles/ml, p<3.96E-05), PEG-SMRwt-CLUpeptide in combination with paclitaxel (7.5×10̂8 particles/mL, p<0.001)and PEG-SMRwt-CLU peptide in combination with cisplatin (3.06×10⁸particles/mL, p<5.37E-05) (FIG. 4 Panel D). By NTA analysis, the size ofthe exosomes was estimated to range between 30 to 47 nm in both breastcancer cell lines.

Finally, Western blot analysis was used to detect exosome proteins incontrol- and peptide-treated cultures. The results of this analysisrevealed the presence of human CD63 and Alix markers in the all exosomesisolated from MCF-7 cells (FIG. 5) and MDA-MB-231 cells (FIG. 6).Control exosomes showed higher expression of human CD63 from MCF-7 cellsand higher expression of Alix from MDA-MB-231 cells.

Example 6: Blocking the SMR-Mortalin Interaction Blocks Exosome Releasein Breast Cancer Cells

A previous study identified the HSP70 family protein, mortalin (encodedby HSPA9) as a binding partner for HIV-1 Nef SMR, and showed thatdisruption of HIV-1 Nef SMR-mortalin binding interfered with exosomerelease (Shelton M N. et al., 2012). To test whether an analogousinteraction accounts for the observed PEG-SMRwt-CLU effect on exosomerelease from breast cancer cells, MCF-7 cells were transfected withantibody to mortalin or antibody to α-tubulin as a control. Theanti-mortalin treated cells were found to be significantly impaired inexosome release as measured by AchE assay (FIG. 7 Panel A) and slightlyless so when measured by NTA assay (FIG. 7 Panel B). The effect oftreatment with anti-mortalin was similar to the effect of treating MCF-7cells with PEG-SMRwt-CLU peptide.

To further validate the significance of this mortalin-mediated processin cancer cells, expression of mortalin protein was knocked down bytransfecting MCF-7 cells with a plasmid construct expressing a mortalinsiRNA. The mortalin siRNAs were found to block exosome secretion asevidenced by AchE assay and membrane fluorescence (N-Rh-PE) assays atall time points tested (FIGS. 8 Panel A and 8 Panel B) in the absence ofany cell toxicity (FIG. 8 Panel C). The exosomes from siRNA-transfectedcells were further assayed for expression of mortalin and the exosomemarker CD63, a tetraspanins by Western blot analysis. The results ofthis analysis showed that expression of both mortalin and CD63 wassignificantly decreased at 48 h on through to 96 h (FIGS. 8 Panel D and8 Panel E).

The above description is for the purpose of teaching the person ofordinary skill in the art how to practice the present disclosure, and itis not intended to detail all those obvious modifications and variationsof it which will become apparent to the skilled worker upon reading thedescription. It is intended, however, that all such obviousmodifications and variations be included within the scope of the presentembodiment, which is defined by the following claims. The claims areintended to cover the claimed components and steps in any sequence thatis effective to meet the objectives there intended, unless the contextspecifically indicates the contrary.

What is claimed is:
 1. A multipartite peptide that inhibits release ofexosomes in a cell, comprising an N-terminal end and a C-terminal endand comprising at least one secretion modifying region (SMR) peptidefrom HIV-1 Nef and at least one Clusterin (Clu)-binding peptide(Clu-BP).
 2. The multipartite peptide of claim 1, wherein the at leastone SMR peptide comprises the amino acid sequence VGFPV (SEQ ID NO: 1)or VGFPVAAVGFPV (SEQ ID NO:2) and the at least one Clu-BP peptide and atleast one Clu-BP peptide selected from the group consisting ofHPLSKHPYWSQP (SEQ ID NO:3), NTYWSQLLHFQT (SEQ ID NO:4) and SHALPLTWSTAA(SEQ ID NO:5).
 3. The multipartite peptide of claim 1, wherein thepeptide comprises an SMR peptide at the N-terminal end and an Clu-BPpeptide at the C-terminal end.
 4. The multipartite peptide of claim 1,wherein the peptide comprises a Clu-BP peptide at the N-terminal end andan SMR peptide at the C-terminal end.
 5. The multipartite peptide ofclaim 1, wherein the peptide comprises at least two SMR peptidesseparated by a spacer peptide, at least two Clu-BP peptides separated bya spacer peptide, or both.
 6. The multipartite peptide of claim 1, thepeptide comprises an amino acid sequence selected from the groupconsisting of VGFPVAAVGFPVHPLSKHPYWSQP (SEQ ID NO:6),VGFPVAAVGFPVAAHPLSKHPYWSQP (SEQ ID NO:7),VGFPVAAVGFPVAAHPLSKHPYWSQPAAHPLSKHPYWSQP (SEQ ID NO:8).
 7. Apolynucleotide encoding the multipartite peptide of claim
 1. 8. Anexpression vector comprising the polynucleotide of claim 7 operablylinked to a regulatory sequence.
 9. A cell comprising the expressionvector of claim
 8. 10. A pharmaceutical composition comprising themultipartite peptide of claim 1 and a pharmaceutically acceptablecarrier.
 11. The pharmaceutical composition of claim 10, wherein theC-terminal end of the multipartite peptide comprises the Clu-BP peptide.12. The pharmaceutical composition of claim 10, wherein the multipartitepeptide is pegylated.
 13. The pharmaceutical composition of claim 10,wherein the multipartite peptide further comprises an endopeptidasecleavage signal.
 14. The pharmaceutical composition of claim 10,wherein: the multipartite peptide comprises the amino acid sequenceVGFPVAAVGFPV (SEQ ID NO:2); the N-terminal end of the multipartitepeptide is pegylated, and the C-terminal end of the multipartite peptidecomprises the Clu-BP peptide.
 15. A method for treating a cancer,comprising: administering to a subject in need of such treatment aneffective amount of the pharmaceutical composition of claim
 10. 16. Themethod of claim 15, wherein the subject has breast cancer.
 17. Themethod of claim 15, further comprising the step of administering to thesubject a chemotherapeutic agent.
 18. The method of claim 17, whereinthe chemotherapeutic agent is paclitaxel or cisplatin.
 19. A method fortreating an infectious disease, comprising: administering to a subjectin need of such treatment an effective amount of the pharmaceuticalcomposition of claim
 10. 20. The method of claim 19, wherein the subjectis infected with HIV-1 or HIV-2.